JP5579181B2 - Pharmaceutical use of Lanosta-8,24-dien-3-ol - Google Patents

Description

  The present invention generally relates to the tetracyclic terpenol family as anti-inflammatory, anti-carcinogenic and / or analgesic agents by inhibition of certain enzymes whose activity is associated with the growth of certain types of cancer. Is related to the pharmaceutical use of Lanosta-8,24-dien-3-ol.

  In the text that follows, the compound euphol, a member of the lanosta-8,24-dien-3-ol family, will often be mentioned, but this is done merely for ease of reference. For this reason, no other lanosta-8,24-dien-3-ol compound is excluded from the present invention.

  Cancer is the name given to a group of more than 100 diseases that have in common the random growth of cells that invade tissues and organs, and it is known as metastasis but can spread to other body regions. It is possible.

  Different types of cancer correspond to different types of somatic cells. For example, because the skin is formed by more than one type of cell, there are several types of skin cancer. If cancer begins in epithelial tissues such as skin or mucous membranes, it is called a carcinoma. If it begins with connective tissue such as bone, muscle or cartilage, it is called a sarcoma. Other characteristics that distinguish one cancer from another are the rate of cell growth and their ability to invade other tissues and organs near or far from its origin.

  Type C protein kinases (PKCs) comprise a group of protein kinases whose functions and regulation are highly conserved. Kinases are also referred to as phosphotransferases, which phosphorylate serine and threonine residues from their substrates, and gene expression, mitosis, cell motility, metabolism and programmed cell death ( It regulates various cell activities including apoptosis. PKC is phosphorylated before it is activated, but this process occurs during cytosol translocation to the plasma membrane. Their activation and cytosolic translocation to the plasma membrane are responsive to transient increases in diacylglycerol (DAG) or to exogenous substances known as phorbol esters typically present in plants Happens.

  The PKC family contains 12 isoforms divided into the following three subdivisions: Normal (calcium-dependent and cPKC activated by DAG and phosphatidylserine), prototype (nPKC, calcium-independent but activated by DAG and phosphatidylserine) and atypical (calcium-independent And aPKC which is activated by phosphatidylserine but not by DAG). In single cells, the isoforms show differences in their distribution before and after translocation to the cell membrane, so the reference states that the function of each isoform associated with a particular cell is membrane and nuclear It suggests that it may be given by differences in the subcellular location of PKC in the cytoplasmic compartment.

  In recent years, several studies have shown a relationship between the development of pathological processes, including rheumatoid arthritis, multiple sclerosis, colitis, and different types of cancer, and disrupted activation of PKC. The hypothesis about PKC involvement in cancer has recently received much attention, especially based on the finding that these enzymes are substrates for phorbol esters, the natural promoters of tumors. Increased or decreased activation of serine-threonine protein kinases, including PKC, or transcription factors modulated by them can cause disordered cell growth and trigger cancer processes. In that sense, many studies have shown that phosphorylation of transcription factors, particularly nuclear factor Kappa B (NF-κB) and activator protein 1 (AP-1), occurs after PKC activation, In turn, it has been shown to modulate the expression of several proteins important for tumor progression, including cyclooxygenase-2 (COX-2). Thus, such intracellular pathways, activation or blockade of natural compounds, can prevent abnormal cell growth and proliferation.

  Phorbol esters are derived from tetracyclic diterpenes, but are thought to be limited to Euphorbiaceae and Thymelaceae plant families. Such compounds are often studied because of their special tumor-promoting and pro-inflammatory effects. The molecular mechanism that regulates the tumor-promoting trigger of phorbol esters is different from the mechanism that triggers inflammatory activity. Its tumor-promoting trigger is thought to be related to their ability to replace DAG in PKC activation, and further stimulates synthesis of RNA and DNA proteins to behave as mitogenic substances and to grow cells It is thought that it is related also to those ability to stimulate. For pro-inflammatory activity, phorbol esters mobilize phospholipids, release arachidonic acid, and cause prostaglandin secretion, resulting in an inflammatory response in the tissue. Topical application of phorbol esters, in particular TPA (tetradecanoyl phorbol-13-acetate), has contributed to understanding the molecular mechanisms involved in the inflammatory process and cancer.

  Several PKC inhibitors were investigated for the treatment of cancer in different phases of preclinical trials. One of them is enzastaurin (LY317615), which shows significant activity when administered orally and efficacy in different cancer models in vitro and in vivo (Journal of Investigative Dermatology (2006) 126, 1641-1647; Cancer Res (2005) 65: 7462-7469; Mol Cancer Ther. (2006) 5: 1783-1789).

  The main problem in the treatment of anti-inflammatory diseases with NSAIDs (non-steroidal anti-inflammatory drugs) and steroidal drugs (corticoids) is their side effects and their total ineffectiveness. The most frequently reported side effects are gastric diseases such as headache, stomach pain, vomiting, diarrhea, gastric ulcer and duodenal ulcer. Corticoids increase blood pressure, astheny and myopathy, peptic ulcers, petechies, erythema, acne, chronic headache, hirsutism, childhood growth inhibition (for long-term treatment), May cause amenorrhea, cataracts and glaucoma, appetite and weight gain, nausea. NSAIDs, a COX-2 option, can also increase the risk of severe thrombotic cardiovascular events, coronary thrombosis and stroke.

  The challenge for effective treatment for cancer is determining the distinction between malignant and normal somatic cells. They share the same origin and are very similar, leading to a lack of recognition on the part of the immune system facing the threat. To date, cancer has been treated by surgery, chemotherapy, radiation therapy and immunotherapy (monoclonal antibody therapy). The choice of treatment depends on the location, extent and stage of the tumor and the general condition of the patient. Complete removal of the tumor without damage to the rest of the body is the primary objective of the procedure, which can sometimes be obtained by surgery, but invades adjacent tissues or propagates to remote sites ( Metastatic) disease propensity often limits its efficacy. The efficacy of chemotherapy is often limited by toxicity to other cells in the body, and radiation therapy can damage normal tissue. In the case of immunotherapy, cancer cells produce a mechanism that escapes the immune response, a phenomenon known as treatment resistance.

Journal of Investigative Dermatology (2006) 126, 1641-1647 Cancer Res (2005) 65: 7462-7469 Mol Cancer Ther. (2006) 5: 1783-1789

  In view of the prior art, the present invention relates to the use of lanosta-8,24-dien-3-ol for the inhibition of enzymes whose activity is related to the growth of cancer cells, in particular PKC. I will provide a. Thus, the present invention further relates to lanosta-8,24-dien-3-ol for use in the effective treatment of tumors, inflammation and / or pain, without significantly accompanying the hitherto known drawbacks.

FIG. 1 shows the difference in ear thickness before and after application of a test compound over time. FIG. 2 shows the absorbance of the three test compositions. White frame: positive control, ie, 2.5 μg / ear TPA (tetradecanoylphorbol-13-acetate) in vehicle containing a 3: 1 acetone: ethanol mixture; black frame: negative control, ie 3: Vehicle: 1 acetone: ethanol mixture; and gray frame: mixture of 100 μg / ear Euphor and 2.5 μg / ear TPA in vehicle containing 3: 1 acetone: ethanol mixture. FIG. 3A is a time vs. frequency graph of the response of each test compound. FIG. 3B is a representation of the area under the curve of FIG. 3A giving a comparative percentage between each test compound. FIG. 4A is a graph of time vs. frequency of response for each test compound. FIG. 4B is a representation of the area under the curve of FIG. 4A giving a comparative percentage between each test compound. FIG. 4C is a representation of the area under the curve of FIG. 4A giving a comparative percentage between each test compound. FIG. 5A is a time vs. frequency graph of the response of each test compound. FIG. 5B is a representation of the area under the curve of FIG. 4A giving a comparative percentage between each test compound. FIG. 5C is a representation of the area under the curve of FIG. 4A giving a comparative percentage between each test compound.

  Suitable lanoster-8,24-dien-3-ols, without excluding any others, euphor, tirucallol and lanosterol, their isomers, derivatives (specifically acetate), solvates Compound or hydrate. Lanosta-8,24-dien-3-ol can be obtained, for example, from Euphorbiaceae plants or by chemical synthesis, but the route is unsuitable for the present invention.

  Thus, in a first aspect, the present invention provides a method for producing a pharmaceutical composition that inhibits increased or decreased activation of serine-threonine protein kinases or transcription factors modulated by them, It relates to the use of 24-dien-3-ol. The compounds of the present invention inhibit the activation of such kinases, specifically PKC (protein kinase C), but the activity is known to be associated with cancer cells. More specifically, transcription factors modulated by these kinases include nuclear factor Kappa B (NF-κB) and / or activator protein 1 (AP-1).

  The lanosta-8,24-dien-3-ol of the present invention and compositions comprising them can be administered to a subject in need of treatment by oral, topical, transdermal, subcutaneous, intraperitoneal, intravenous, infiltration, inhalation. It can be administered by any appropriate method, including transdermal, transmucosal, intramuscular, intrapulmonary, vaginal, rectal, intraocular and sublingual. Specifically suitable administration methods in the present invention are local and systemic (infiltration, oral, spray inhalation, transdermal) administration. The lanoster-8,24-dien-3-ol of the present invention can be included in a sustained or controlled release composition. Known adjuvants and excipients can be utilized in such compositions. A discussion of pharmaceutical dosage forms useful in the compositions according to the present invention can be found in the publication Remington's Pharmaceutical Sciences, Mack Publishing, 1965-1990.

  Without excluding any other members of the family, suitable lanosta-8,24-dien-3-ols may be one or more of Euphor (RN514-47-6), Tilcarolol (RN514-46- 5) and lanosterol (RN79-63-0), more specifically Euphor.

  In another aspect, the present invention provides lanosta-8,24-dien-3-ol or the same for the manufacture of a pharmaceutical composition for the treatment of tumors and / or inflammation and / or pain (nociceptive response). It relates to the use of the composition comprising.

  The compositions of the present invention can be used as solids, liquids or semi-liquids, tablets, capsules, pills, powders, granules, suspensions, emulsions, dispersions and any other useful known pharmaceutically. It can be administered to a patient in an acceptable form. The compositions will contain additional active agents, such as antibiotics, depending on the desired action. For oral administration as tablets or capsules (both soft and hard capsules), lanoster-8,24-dien-3-ol is added to lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, diphosphate. Can be combined with pharmaceutically acceptable inert vehicles such as calcium, calcium phosphate, mannitol, sorbitol and the like; for oral administration in liquid form, lanosta-8,24-dien-3-ol Can be combined with ethanol, glycerol, water and the like. If desired or necessary, flocculants, lubricants, disintegrants, colorants and fragrances can be added to the mixture. Common flocculants are natural or synthetic gums such as glucose, [β] -lactose, corn sweetener, gum arabic, tragacanth, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride. Disintegrants include starch, methylcellulose, agar, bentonite, xanthan gum and the like.

  The compositions of the present invention can further be administered as liposomes or coupled to a soluble polymer such as a vehicle.

  Liquid dosage forms for oral administration can contain coloring and sweetening agents to increase acceptance by the patient. Vehicles acceptable for water dosage forms are water, suitable oils, saline solutions, aqueous dextrose, other sugar solutions, and glycols such as propylene glycol or polyethylene glycol, phosphate buffers.

  In yet another aspect, the invention relates to the appearance of inflammation and / or cancer and / or pain associated with a perturbed activation of serine-threonine protein kinase, more specifically PKC A method of medical treatment for those physical conditions when affecting presence, to a pharmacologically effective amount of a pharmacologically acceptable carrier or excipient for treatment of the condition to the mammal It relates to a method comprising administration in.

  The following examples are specific embodiments of the present invention, but they are in no way intended to limit it beyond what is presented in the appended claims.

  In all the following examples, 20-30 g male mice were placed in a filtered air draft cage with a controlled temperature (22 ± 2 ° C.) and humidity (50-60%), 12 hours light / 12 hours dark cycle. Maintained free access to water and food. The test animals were kept in the laboratory for adaptation for at least one hour prior to the pharmacological test conducted at 8: 00-17: 00.

  In all the examples below, statistical analysis was performed with Graph Pad Prism® 5.0 software.

Example 1
Characterization of the stimulatory effects of certain compounds was performed by comparing the results of subsequent topical treatment of mouse ears.

-Positive control, ie, 2.5 μg / ear TPA (tetradecanoylphorbol-13-acetate) in a vehicle containing a 3: 1 acetone: ethanol mixture;
A negative control, ie a vehicle that is a 3: 1 acetone: ethanol mixture;
-100 [mu] g / ear compound Euphor of the invention in a vehicle containing a 3: 1 acetone: ethanol mixture;
A mixture of 100 μg / ear Euphor and 2.5 μg / ear TPA in a vehicle containing a 3: 1 acetone: ethanol mixture.

  The above composition was topically applied to the inner surface of the ear of a group of 5 member mice. Ear thickness was measured using a digital micrometer before and after exposure to the test composition and the response was expressed as mμ. FIG. I shows the difference in ear thickness over time before and after application of the test compound.

  The results are shown in FIG.

  As can be seen, the inhibitory effect obtained with Euphor was significant up to 24 hours after treatment, indicating an important pharmacokinetic effect on inflammation.

Example 2
This was an assessment of the effect of the compounds of the present invention on PKC activity on mouse skin. The test animals were given the following 100 μg / ear composition.

-Positive control, ie, 2.5 μg / ear TPA (tetradecanoylphorbol-13-acetate) in a vehicle containing a 3: 1 acetone: ethanol mixture;
A negative control, ie a vehicle that is a 3: 1 acetone: ethanol mixture;
A mixture of 100 μg / ear Euphor and 2.5 μg / ear TPA in a vehicle containing a 3: 1 acetone: ethanol mixture.

  The above composition was topically applied to the inner surface of the ear of a group of 5 member mice. PKC activity was measured using ELISA (solid phase enzyme immunoassay) before and after exposure to the test composition. FIG. 2 shows the absorbance of the three test compositions listed above.

  As can be seen, TPA promotes a significant increase in PKC activity for the vehicle-treated group, but treatment with Euphor significantly reduces the increase in PKC activity induced by TPA.

Examples 3, 4 and 5- Nociceptive Nociceptive mechanical thresholds were assessed as the frequency of paw withdrawal responses after 10 applications of Von Frey filament (VHF, Stoelting, Chicago, USA). Test animals were individually placed on an elevated wire mesh platform in a 9x7x11 cm clear acrylic chamber to allow access to the sole of the foot. Von Frey filament was applied to the right hind paw and the following criteria were watched: (1) Apply perpendicular to the bottom with sufficient pressure to bend the filament to ensure total pressure; (2) Test animals were evaluated when all four legs were on the wire mesh; (3) Paw The withdrawal response was considered as when the animal fully retracted its paw from the wire mesh; (4) Each animal was stimulated 10 times continuously with a 1 second duration of stimulation; (5) each Paw withdrawal was considered a 10% response and 10 withdrawals corresponded to a 100% response.

Example 3 Carragenan-Induced Inflammatory Nociception For triggering of inflammatory pain, each test animal received 20 μl intraplantar carrageenan injection (300 μg / foot) in the right hind paw. Test animals treated with 0.9% (20 μl / paw) PBS (phosphate buffered saline) solution were used as controls. Carrageenan dosage results in edema, nociception and significant size increase in the injected paw.

  Test animals were treated orally with Euphor (30 μg / kg) one hour prior to carrageenan injection. Test animals treated with a subcutaneous injection of 0.5 mg / kg dexamethasone 4 hours prior to carrageenan injection were used as positive controls. Hypernociception was assessed with 0.6 g Von Frey filament every hour for 8 hours, as well as 24 and 48 hours after carrageenan injection.

  The results are shown in FIGS. 3A and 3B, where 3A is a graph of time vs. frequency of response for each test compound, and 3B is the area under the curve of 3A giving a comparative percentage between them. It is a display.

  As can be seen in the figures, acute treatment with Euphor significantly reduced carrageenan-induced inflammatory mechanical nociception. Euphor results in a reduced nociceptive response similar to the control group treated with dexamethasone.

Example 4-Persistent Inflammatory Nociception Induced by CFA (Complete Freund's Adjuvant) 25 μl of CFA, a dose that results in high nociception and increased size of the injected foot, is injected into the sole of the subject animal (Neuropharmacology, 41: 1006-1012, 2001; Anesth Analg., 101: 1763-1769, 2005).

  The test animals were treated orally with 30 mg / kg Euphor or 70 mg / kg gabapentin (positive control) one hour prior to CFA injection. High mechanical nociception by stimulation with 0,6 g Von Frey filament, at time intervals of 1, 2, 4, 6, 8, 24 and 48 hours after CFA injection and nociceptive response Was measured until reconfirmed. Thereafter, that is, on the third day, long-term treatment was started and long-term treatment with Euphor was evaluated. In that regard, test animals were given 30 mg / kg Euphorol orally daily for 5 days, and hypernociception was assessed once a day for 4 hours after the first dose. Mechanical nociception was assessed until the painful response returned. Thereafter, once daily treatment was resumed for 5 days to assess the development of tolerance to the compound and high nociception was assessed until the nociceptive response returned.

  The results are shown in FIGS. 4A, 4B and 4C, where 4A is a graph of the time vs. frequency of the response of each test compound, and 4B and 4C are 4A's giving a comparative percentage between them. It is a display of the area under the curve.

  As can be seen in FIGS. 4A and 4B, acute treatment with Euphor significantly reduced CFA-induced mechanical nociception near the effect obtained with gabapentin.

  When Euphor is administered once a day for 5 days, CFA-induced nociceptive inhibition is observed for 6 days following treatment. The mechanical nociception decreased again at the beginning of the long-term treatment, as can be seen by the curves from day 3 to day 24 after CFA injection. Similar inhibition was observed with prolonged treatment with gabapentin.

Example 5 Neuropathic Pain Induced by Partial Stenosis of the Sciatic Nerve The procedure used here is described for rats (Pain, 43: 205-218, 1990) and modified for mice (Pain, 76 : 215-222, 1998) and those standardized by Bortolanza et al (Eur J Pharmacol., 453: 203-208, 2002). Mice were anesthetized with 7% chloral hydrate (0.6% ml / kg in plantar). Next, the sciatic nerve was exposed on the thigh near the sciatic nerve trifurcation and the dorsal portion 3/1 to 2/1 was tied with suture filament # 8. In the sham operation group, the sciatic nerve was exposed without being tied. On the fourth day after surgery, one group of test animals was orally treated with 30 mg / kg Euphor and another group was orally treated with 70 mg / kg gabapentin as a positive control. At predetermined times after treatment (1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours and 48 hours), mechanical thresholds were evaluated by stimulation with 0.6 g Von Frey filament. In order to assess the effects of long-term treatment, the subjects were administered euphoric daily for 6 to 5 days and mechanical nociception once a day, 4 hours after the first administration, in the control group. And evaluated until nociceptive responses returned. Thereafter, the long-term treatment protocol was repeated to evaluate the possible development of resistance to the compound.

  The results can be seen in FIGS. 5A, 5B and 5C, where 5A is a graph of the time vs. frequency of each test compound response, and 5B and 5C give the comparative percentage between them It is a display of the area under the curve of 4A.

  As can be seen in FIGS. 5A and 5B, acute treatment with Euphor or gabapentin significantly inhibited the mechanical nociceptive response induced by partial stenosis of the sciatic nerve.

  Similarly, once-daily long-term treatment with Euphor or gabapentin significantly inhibits mechanical nociception induced by partial stenosis of the sciatic nerve by returning to nociceptive response on day 10 from treatment did.

  With the help of the contents and examples set forth herein, those skilled in the art will be able to use the same function to obtain similar results without departing from the scope of the invention as defined in the claims. Can be reproduced evenly.

Claims (5)

Of lanosta-8,24-dien-3-ol for the manufacture of a pharmaceutical composition for the treatment of pain that inhibits the increased or decreased activation of protein kinase C or transcription factors modulated by them use. Use according to claim 1, wherein the transcription factor is nuclear factor Kappa B (NF-κB) and / or activator protein 1 (AP-1). The Ranosuta -8,24- dien-3-ol, Yoo Horu (euphol), Chirukaroru (tirucallol) and lanosterol one or more, their isomers, derivatives, solvates or hydrates, claim 2. Use according to 1, wherein the derivative of Lanosta-8,24-dien-3-ol is acetate. The use according to claim 1, wherein the lanosta-8,24-dien-3-ol is euphor. A pharmaceutical composition comprising lanosta-8,24-dien-3-ol for use in the treatment of pain.

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